The precise mechanisms by which starch is synthesised and degraded in plants are unknown, despite the isolation and characterisation of a number of enzymes that are presumed to be involved in the process.
Starch is accumulated in the chloroplasts of leaves during the day and is used to supply the needs of the plant for energy and biosynthesis during the night. The mode by which this so-called transient starch is mobilised is not fully understood, but must involve the co-ordinated regulation of synthetic and degradative enzyme activities. In leaf tissues the main degradation pathway is thought to involve phosphorolytic and hydrolytic activities, especially xcex1-glucosidase (E.C. 3.2.1.3) (Nielson and Stitt, 1997).
Starch is also accumulated in the amyloplasts in storage organs such as seeds, fruit and tubers. In this case starch is stored over longer periods of time and mobilisation of the starch is accompanied by degeneration of the storage organ tissues and increases in amylolytic and phosphorolytic activities. However, there is evidence to suggest that turnover of starch is also occurring in the amyloplasts of the storage organ (Sweetlove et al, 1996). This again requires the co-ordinated regulation of the synthetic and degradative enzyme activities.
Chloroplasts and amyloplasts are both derived from proplastids and therefore have many characteristics in common besides being the site of starch synthesis in leaves and storage organs respectively; chloroplasts can be converted to amyloplasts and other types of plastid (Thomson and Whatley, 1980).
Starch is a mixture of two polysaccharides: amylose which is a linear chain of glucosyl units linked by xcex1-1,4-glycosidic bonds; and amylopectin which is made up of many linear chains of xcex1-1,4-polyglucans which are joined together by xcex1-1,6 glycosidic bonds.
Enzymes involved in the synthesis of starch are ADPG pyrophosphorylase (E.C. 2.7.7.21), starch synthase (E.C. 2.4.1.21) and branching enzyme (E.C. 2.4.1.18). ADPG pyrophosphorylase is responsible for supplying the substrate ADPG, this molecule serving as the donor of glucose monomers which are linked together by the concerted action of starch synthases (xcex1-1,4 bonds) and branching enzymes (xcex1-1,6 bonds).
It is thought that the insoluble, crystalline structure of starch grains is formed by the close packing of the extended helical, branched amylopectin molecules, with the linear amylose molecules filling any spaces.
A range of starch-degrading enzyme activities has been reported including xcex1-amylase (E.C. 3.2.1.1), isoamylase (E.C. 3.2.1.68), xcex2-amylase (E.C. 3.2.1.2), xcex1-glucosidase (E.C. 3.2.1.3), starch phosphorylase (E.C. 2.4.1.1) and disproportionating enzyme (E.C. 2.4.1.25). Many of these enzyme activities exist in multiple forms in plants and some are thought to be involved in the synthesis of starch. All probably take part, to some extent, in the starch mobilisation process, however their exact roles and possible interactions are yet to be determined. The difficulties in attributing roles for the different enzymes is best exemplified by reference to two of the enzyme activities which are thought to. be the major contributors to starch breakdown in plants: starch phosphorylase and amylase.
Starch phosphorylase catalyses the reversible release of glucose-i-phosphate from xcex1-1,4-glucans. Two forms of starch phosphorylase are found in plant tissues: Pho1, or the L-type, is located inside plastids and has a high affinity towards maltodextrins; Pho2, or the H-type, is cytosolic and has high affinity to large, highly branched polyglucans such as glycogen. Although the plastidic Phol enzyme would be a likely candidate to be involved in the mobilisation of starch, antisense inhibition of the leaf enzyme activity had no effect on the starch accumulation in leaves of transgenic potato plants (Sonnewald et al., 1995). In another study, antisense inhibition of the cytoplasmic Pho2 had an influence on the sprouting behaviour of transgenic potato tubers, but had no effect on the starch accumulation and degradation (Duwenig et al., 1997).
There are two major groups of amylase both of which hydrolyse xcex1-1,4-glucosidic linkages in amylose and amylopectin: xcex1-amylase acts randomly on non-terminal linkages, whereas xcex2-amylase acts to release maltose units starting from the non-reducing end of the polyglucan chain. The subcellular location of xcex1-amylase in the apoplastic space of plant cells is thought to reflect the fact that the enzyme is normally secreted. However, in a number of plants such as rice (Chen, et al., 1994) and sugar beet (Li, et al., 1992) the enzyme is also located inside chloroplasts and amyloplasts, despite the finding that the signal sequences at the amino-terminus of a number of xcex1-amylase proteins are characteristic for translocation of protein across the ER membrane rather than the plastid membrane (Chen et al, 1994). In a study where the promoter and signal sequence of a rice xcex1-amylase gene was fused to the bacterial GUS gene and introduced into rice, tobacco and potato using Agrobacterium-mediated transformation (Chan et al., 1994), it was demonstrated that the expressed GUS fusion protein was first transported to the endoplasmic reticulum and then exported into the culture medium of suspension cultures made from transgenic cells. It has been shown in a number of studies that xcex1-amylase will degrade native starch molecules.
In contrast, in vitro studies have shown that xcex2-amylase will not degrade native starch granules without prior digestion of the granule with other enzymes. Mutants of rye (Daussant et al., 1981) and soybean (Hildebrand and Hymowitz, 1981) that lack active xcex2-amylase or contain only traces of activity, respectively, apparently show normal growth and development. In addition, transgenic Arabidopsis plants in which the levels Of xcex2-amylase have been greatly reduced, do not show severe growth defects (Mita et al., 1997). Attempts to define the precise physiological role of xcex2-amylases in plants have been hampered by inconclusive data concerning subcellular location. Although one study (Kakefuda et al., 1986) reported the presence of two, xcex2-amylases in pea chloroplasts, most studies involving species such as Vicia faba, barley, wheat, soybean, sweet potato and pea have concluded that most, if not all, xcex2-amylase activity is extrachloroplastic (Nakamura et al., 1991). This view is supported by the fact that all xcex2-amylase genes cloned to date encode proteins that lack amino-terminal chloroplast transit peptide sequences.
In cereals, three types of xcex2-amylase have been described: an endosperm-specific form that accumulates during caryopsis maturation; a form that is synthesised de novo in aleurone cells of rice and maize during germination (Wang et al., 1996; 1997); and a xcex2-amylase which is ubiquitous in vegetative organs. In Arabidopsis, the ubiquitous form accounts for approximately 80% of the total starch-degrading activity of rosette leaves. In common with all other xcex2-amylase genes cloned to date, the gene for the ubiquitous Arabidopsis xcex2-amylase does not encode a protein with a subcellular targeting signal, thus the enzyme is likely to be located in the cytosol.
The findings from a number of studies that the degradative activities can be removed without an adverse effect on the viability of the plant, plus the subcellular location of starch degrading enzymes outside the plastid, is surprising. The apparent absence of a plastid-localised xcex2-amylase activity is especially surprising in light of the fact that the expected major end-product of xcex2-amylase activity, namely maltose, has been identified as a product of starch degradation in isolated chloroplasts (Peavey et al., 1977). More recently, it has been shown that both glucose and maltose are exported from isolated cauliflower bud amyloplasts during the process of starch mobilisation (Neuhaus et al., 1995).
The ability to manipulate the amount of starch in the plastids of leaves or storage organs would be of high benefit to various industrial processes which utilise plant starches. For example, in an attempt to increase the starch content of potato tubers, it has been shown previously that when E. coli ADPG PPase glgC16 is overexpressed in transgenic potato tubers, there is an increase in flux of carbon into starch but there is only a small increase in net accumulation of starch (Sweetlove et al., 1997). Analysis of enzyme activities in the overexpressing lines showed that, apart from the alteration in ADPG PPase, the activity of amylase, specifically xcex2-amylase was also altered. This data suggests that the accumulation of starch in tubers overexpressing glgC16 protein is prevented by the breakdown of the newly synthesised starch, i.e. the starch is being turned over.
In another example, the availability of starch during the malting process is closely correlated with the types and amounts of degradative enzyme activities in the plant, specifically the storage organs. An increase in the degradative capacity of the crop would make the malting of cereal grain or the conversion of starch from tubers, or other storage organs, to alcohol more efficient and productive.
The type of starch present in the storage organ depends on the forms and activities of the ADPG pyrophosphorylase, starch synthase, branching enzyme and the degradative enzymes present. The interactions between the various enzymes will also be important.
There is considerable interest in creating novel starches in planta as this will reduce the costs of processing and modification of the starch before use in a variety of industries such as food, paper, pharmaceuticals, glue, oil and textiles. The following examples show how starch hydrolytic activity can be important in altering the structure of starch in vivo.
It has been shown that, in maize kernels, the sugaryl mutation causes the absence of a debranching enzyme which hydrolises xcex1-1,6-glycosyl linkages of starch (James et al., 1995). The mutation results in the decreased concentration of amylopectin and accumulation of the highly branched glucopolysaccharide, phytoglycogen.
It has been shown that in pea, short oligosaccharide molecules, starting with maltose and adding successive glucose units up to maltoheptose, specifically stimulate the activity of granule bound starch synthase I (GBSSI) (Denyer et al., 1996) which is generally accepted to be the major enzyme responsible for the synthesis of amylose (e.g. van der Leij et al., 1991; Hylton et al., 1995; Ainsworth et al. , 1993). The manipulation of GBSSI activity by controlling the supply of malto-oligosaccharides is the subject of a recent patent (WO 97/16554) and suggests that an increase in the concentration of malto-oligosaccharides, and thus an increase in the ratio of amylose to amylopectin in the starch, can be brought about by the introduction of degradative enzymes namely xcex1-amylase, xcex2-amylase, disproportionating enzyme, debranching enzyme and starch phosphorylase. Patent WO 97/16554 also states that genes for plastidial isoforms of these enzymes have been cloned. However, as discussed above, no xcex2-amylase genes isolated to date encode a xcex2-amylase enzyme with a protein targeting sequence and, in addition, there is doubt that xcex1-amylases are originally targeted to plastids (Chen et al., 1994; Chan et al., 1994). Later in WO 97/16554, reference is made to the engineering of a suitable xcex2-amylase CDNA sequence to add a plastid targeting sequence.
In addition to the industrial uses for starch in the storage organs, the amount of starch in the leaf has significant importance for the agronomy of a crop. Starch is synthesised in the leaf during daylight from the carbon fixed during photosynthesis. The starch is stored in the chloroplast and is broken down at night to become a source of energy and intermediates for metabolism in the plant. By which mechanisms the source-sink relationship is controlled are unknown at present, however, it is clear that manipulation of the amount and availability of the starch in leaf plastids will have a profound influence on plant productivity (biomass and yield).
The amount of starch in the leaf will also be important for those crops where the leaf is the major plant commodity, for example tobacco. It is known that starch content has an influence on the eventual flavour of tobacco when smoked. Provision of a means to manipulate the level of starch in tobacco leaves could be of interest to the tobacco industry.
We describe here, for the first time, the isolation of a cDNA encoding a novel xcex2-amylase enzyme which is targeted to plastids (henceforth known as chloroplast targeted (ct) xcex2-amylase), by a novel targeting sequence. The isolation of this entire coding sequence is surprising, as it has generally been thought that xcex2-amylase would only take part in the hydrolysis of starch once smaller polyglucan fragments had been released, either by translocation or through breakdown of the membrane, from the plastid into the cytoplasm. Location of the enzyme in plastids opens up the unforeseen possibility that ct xcex2-amylase is involved in the degradation of transient starch located in chloroplasts and storage starch located in amyloplasts.
The similarity of characteristics between chloroplasts and amyloplasts (Thomson and Whatley, 1980) is of relevance to the current invention, as it has been shown that the transit peptides from chloroplast-targeted polypeptides can import heterologous polypeptides into amyloplasts and vice versa. For example, the transit peptide from the maize granule bound starch synthase enzyme when fused to the E. coli xcex2-glucuronidase (GUS) protein will import the GUS protein not only into amyloplasts but also into chloroplasts (Klosgen and Weil, 1991).
In addition, we show that expression of the ct-Bmy gene in Arabidopsis and the expression of ct-Bmy promoter:GUS fusions in transgenic tobacco can be regulated independently by both light and sucrose. This is surprising in view of the tightly coupled light and sugar induction responses of ATxcex2-Amy of Arabidopsis (Mita et al, 1995).
The present invention provides a nucleic acid sequence known herein as SEQ. ID. No. 1 and being from 1-294 nucleotides and having therewithin a sequence capable of targeting a further coding sequence to a plant plastid, or sequences being at least 65% or more homologous with the disclosed sequence SEQ. ID. No. 1 and having the same targeting ability.
Preferably the nucleic acid sequence encodes about 94 and more preferably about 85 amino acid residues.
The present invention also provides a nucleic acid sequence known herein as SEQ. ID. No. 2 and being from 1-1642 nucleotides and having therewithin a sequence capable of encoding xcex2-amylase, or sequences being at least 65% or more homologous with the disclosed sequence within SEQ. ID. No. 2 and having the same encoding ability.
The present invention also provides a nucleic acid sequence known herein as SEQ. ID. No. 3 and being from 1-1953 nucleotides and having therewithin a sequence capable of encoding chloroplast targeted xcex2-amylase, or sequences being at least 65% or more homologous with the disclosed sequence within SEQ. ID. No. 3 and having the same encoding ability.
Homologous sequences also include those sequences which hybridise to SEQ. ID. No. 1, SEQ. ID. No. 2 or SEQ. ID. No. 3 under medium stringency conditions (washing at 2xc3x97SSC at 65xc2x0 C.).
Preferably the nucleic acid sequence is an mRNA or cDNA sequence, although it may be genomic DNA.
The present invention also provides a method of increasing or decreasing in a plant the activity of an enzyme in the pathway of starch biosynthesis or degradation, the method comprising the steps of stably incorporating into a plant genome a chimaeric gene comprising a nucleic acid sequence encoding a plastid targeting sequence and a coding sequence for an enzyme in the starch biosynthetic or degradative pathway, and regenerating a plant having an altered genome.
The present invention also provides a method of targeting proteins or enzymes to a plant plastid, the method comprising the steps of stably incorporating into a plant genome a chimaeric gene comprising a nucleic acid sequence encoding a plastid targeting sequence and a coding sequence for a protein or an enzyme, and regenerating a plant having an altered genome, the protein or enzyme being one or more in the pathway of the following group: lipid synthesis, photosynthesis, amino acid metabolism, nitrogen fixation, carbon fixation or synthesis of carbohydrate polymers; or being able to confer a characteristic to the plant, the characteristic being selected from one or more of the following group: herbicide resistance and pest resistance, for example, including fungal, bacterial or viral resistance.
The present invention also provides plants having therein a chimaeric gene comprising a promoter, a nucleic acid coding sequence encoding the plastid targeting sequence, the sequence being capable of targeting a coding sequence of an enzyme in the starch biosynthetic or degradative pathway to a plant plastid, and a terminator.
The present invention further provides a nucleic acid sequence capable of directing expression of a product encoded by a coding sequence which is operably linked thereto, said nucleic acid sequence being known herein as SEQ.ID. No. 8, or being at least 65% homologous therewith and having substantially the same function thereas, and said nucleic acid sequence being responsive to stimulus, the level of expression of said product being variable in response to the stimulus applied to said nucleic acid sequence.
The present invention further provides a method of varying the level of expression of a product encoded by a coding sequence operably linked to a nucleic acid sequence capable of directing expression of said product in a plant, said method comprising the steps of stably incorporating into a plant genome a chimaeric gene comprising a nucleic acid sequence capable of directing expression of a product encoded by a coding sequence that is operably linked thereto, said nucleic acid sequence having substantially the sequence of SEQ.ID. No. 8 or being at least 65% homologous therewith and having substantially the same function thereas, and being responsive to stimulus.
Preferably the stimulus is the presence or absence of light and/or varying levels of sugar. Alternatively the stimulus is a stimulus which is developmentally controlled.
Advantageously the sugar is one or more of sucrose or glucose.
Preferably the sugar is sucrose.
Advantageously the inducible promoter, or nucleic acid sequence capable of directing expression of said product in a plant, is operable under conditions when there is no light but sugar is present, or when there is no sugar but light is present. The tissue of a plant where no light but sugar is present may suitably be underground organs or sink organs. Underground organs may be, for example, tubers, rhizomes or roots, whereas other sink organs may be young leaves or seeds.
The tissue of a plant where no sugar but light is present may be older leaves (where no sugar is transported), flower parts or germinating seeds.
Constructs and chimaeric genes having the DNA structural features described above are also aspects of the invention.
Plant cells containing a chimaeric gene comprising a nucleic acid sequence encoding a plastid targeting sequence hereinabove described and a nucleic acid coding sequence of an enzyme in the starch biosynthetic or degradative pathway, or a chimaeric gene comprising a nucleic acid sequence capable of directing expression of a further coding sequence, or a chimaeric gene comprising a nucleic acid sequence hereinabove. described that is responsive to stimulus and a coding sequence, the level of expression of said coding sequence being variable in response to the stimulus applied to said nucleic acid sequence are also an aspect of this invention, as is the seed of the transformed plant containing one or more chimaeric genes according to the invention.
Advantageously the plastid targeting sequence is the sequence SEQ. ID. No. 1.
In a first aspect of the invention the above method may be used to alter the metabolism of a leaf such that starch is accumulated therein or mobilised therefrom, this process altering the source-sink relationships within the plant as a whole. Such may be achieved by providing the targeting sequence and a nucleic acid coding sequence of an enzyme in the starch biosynthesis or degradative pathway under the direction of a suitable promoter. Suitable promoter selection would result in plants with increased or decreased levels of starch in the leaves which might be useful, for example, in the tobacco industry; or alternatively would result in changes in yield of starch in various other plant tissues such as tubers, fruit and roots following modification of the source-sink relationships of the plant.
In this embodiment of the invention a suitable promoter would direct expression of the plastid targeting sequence and the coding sequence of an enzyme in the starch biosynthetic or degradative pathway throughout the whole plant, so called constitutive expression, or specifically to the leaves. These changes will have a profound effect such that the starch content and/or the yield of the organs of the plant would be significantly altered.
A preferred promoter capable of directing expression throughout all plant tissues is the full or truncated promoter taken from cauliflower mosaic virus 35S gene. For storage organ expression, preferred promoters can be taken from the high molecular weight glutenin gene, the xcex1, xcex2-gliadin gene, the hordein gene and the patatin gene. For leaf expression, preferred promoters can be taken from the gene for the small subunit of ribulose bisphosphate carboxylase or the pea plastocyanin gene. One skilled in the art will recognise other suitable promoters, for example the nopaline synthase promoter for constitutive expression and the chlorophyll a/b binding protein promoter for specific leaf expression.
The coding sequence, or parts thereof, for the enzyme in the starch biosynthetic or degradative pathway may be arranged in the normal reading frame direction, i.e. sense, or in the reverse reading frame direction, i.e. antisense. Up or down regulation of the activity of the enzyme in a plant using sense, antisense or cosuppression technology (the latter as described by DNAP in their European Patents Nos. 0465572 and 0647715) may be used to achieve alteration in the starch of the plant.
In a second aspect of the invention the inventive method may also be used to alter the metabolism of starch in storage organs such that starch content is increased and/or the starch is provided in a suitable form as required for the purposes of particular industrial processes. Such processes including paper making; manufacture of pharmaceuticals, textiles, dyes and building products; provision of baking, dairy and snack food products; making canned, dried or instant foods; malting of grain and production of syrups and alcohol.
In the first or second aspect of the method the enzyme selected for use in the chimaeric gene of the methods may be one from the starch degradative pathway, i.e. a starch degrading enzyme. Advantageously, the chimaeric gene comprises a chloroplast targeted xcex2-amylase (hereinafter known as ct xcex2-amylase), and more preferably comprises ct xcex2-amylase derived from Arabidopsis thaliana, (hereinafter known as At ct xcex2-amylase), see SEQ. ID. No. 3. Sequences homologous to At ct xcex2-amylase which may be derivable from other plant sources such as potato, tobacco, wheat, maize and barley may also be used. Standard methods of cloning by hybridisation or polymerase chain reaction (PCR) techniques may be used to isolate sequences from such organisms: for example molecular cloning techniques such as those described by Sambrook et al. (1989) and the PCR techniques described by Innes et al. (1990). Other starch degrading enzymes, the coding sequence of one or more of which would be suitable for use with the plastid targeting sequence, include xcex1-amylase, disproportionating enzyme, debranching enzyme, starch phosphorylase, xcex1-glucosidase and non-plastidic xcex2-amylase.
In the second aspect of the inventive method preferred promoters which would direct expression to the storage organs of plants could be selected, for example, from the genes from the following list: the gene for high molecular weight glutenin of wheat endosperm; the gene for xcex1,xcex2-gliadin of wheat endosperm; the hordein gene of barley endosperm; or the gene for patatin from potato tubers. Other suitable promoters are known to those skilled in the art.
In either aspect of the invention, the alteration of tissue metabolism or alteration of starch type or characteristics may be made stimulus responsive, i.e. inducible, by virtue of use of the inducible promoter described herein (SEQ. ID. No. 8). For example, the light inducibility aspect of the inducible promoter could be used to manipulate seed set by inducing a gene such as Barnase (as exemplified in Patent WO 98/10081) to affect pollen development, or to affect non-light responsive genes in otherwise light-dependant processes such as fruit ripening or seed germination. The light inducible promoter could also be used to turn on genes which affect secondary metabolite production in leaves, for example alkaloid production. Light inducible promoters may also be used to manipulate starch biosynthetic enzyme genes in leaves or other photosynthetic tissue, or for example in turning on genes after removal of tubers, for example, from storage in darkness. The sugar-inducibility aspect of the inducible promoter could be used to regulate genes in, for example, developing tuber or other non-photosynthetic tissue such as genes for pest resistance and/or genes which might affect the quality of the post-harvest crop. For potatoes, resistance genes to blight, blackleg and dry rot would be particularly of benefit and could be most advantageously cloned into recombinant genes with sugar inducible promoters. Alternatively, the sugar inducibility aspect of the inducible promoter could be used to drive the expression of genes for selectable markers in the tissue culture process.
One skilled in the art can readily delineate the sugar inducible responsive element from SEQ. ID. No. 8 and/or the light inducible responsive element by using well known techniques, such as deletion studies. Pwee and Gray (1993) describe such a deletion study within the pea plastocyanin gene using a marker gene in order to determine operative regions thereof.
Methods described herein or in, for example, laboratory manuals by Sambrook et al (1989) and Gelvin and Stanton (1995) for cloning gene sequences and inserting them into appropriate carriers (vectors or plasmids etc.) are techniques well known to the skilled man for putting such concepts into effect. The chimaeric gene or genes as described above may be introduced on their own, or be accompanied by one or more other chimaeric genes, such as one or more of the other genes described above. In the case of the above described embodiments utilising a first chimaeric gene encoding an enzyme of the starch degradative pathway, the second chimaeric gene may, for example, comprise a nucleic acid sequence encoding an enzyme from the starch biosynthetic pathway also under the direction of a suitable promoter and a suitable terminator. The promoter and/or terminator of the second chimaeric gene may be the same as or different from the promoter and/or terminator of the first chimaeric gene. Suitable sequences encoding enzymes from the starch biosynthetic pathway are the nucleic acid sequences for sucrose synthase, ADPG pyrophosphorylase, starch synthase, and may also include branching enzyme, xcex1-amylase, isoamylase, non-plastidic xcex2-amylase, xcex1-glucosidase, starch phosphorylase and disproportionating enzyme.
Methods for the introduction of more than one chimaeric gene into a plant have been described and comprise the construction of a binary vector with the chimaeric genes joined together in one nucleic acid molecule; cotransformation using two or more different Agrobacterium cells, for example, with different binary vectors containing different chimaeric genes therein; or the transformation of a plant which already has a chimaeric gene with a second, different chimaeric gene, i.e. retransformation. In the latter case, the method of selection of transgenic plants after the introduction of the second chimaeric gene must be different from the selection method used for the introduction of the first chimaeric gene. Suitable selectable markers would include those for hygromycin, kanamycin, sulphonamide and Basta resistance. Biological methods such as crossing two plants, each plant containing a single chimaeric gene can also be used.
Use of two chimaeric gene constructs could be made in order to alter the starch content of an already transformed plant which shows a significant increase in a first enzyme activity and a consequent change in the synthesis of starch.
Thus, the present invention further provides a method of altering in a transgenic plant, which plant already shows an increase or decrease in an enzyme activity as a result of genetic transformation, a further enzyme in order to up or down regulate said further enzyme and thereby increase or decrease the amount of starch produced by the retransformed plant.
Advantageously the first transformed plant is a plant having an increased enzyme activity in the starch biosynthetic pathway. An example of an attempt to increase the starch content of a plant is a transgenic potato transformed with the gene for ADPG-PPase, for example glgC16 (see for example, WO 91/19806). The amount of starch increase in such a plant has been relatively small. This first transformed plant is advantageously retransformed with a chimaeric gene for a starch degrading enzyme, suitably comprising, for example, At ct xcex2-amylase. The glgC16 protein is expressed in the first transformed tubers and results in increased ADPG-PPase activity and an increase in flux of carbon to starch. Advantageously, the expression of the chimaeric At ct xcex2-amylase gene, or parts thereof, in the retransformed tubers results in down regulation of the ct xcex2-amylase activity, i.e. cosuppression or antisense technology, thus providing for an increase in starch accumulation.
Preferably the expression of the second enzyme is directed to tubers. A suitable promoter to direct the expression of the At ct xcex2-amylase chimaeric gene in tubers is the promoter from the gene for patatin.
The first transformed potato plant expressing glgC16 is kanamycin resistant, therefore the binary vector construct for the At ct xcex2-amylase chimaeric gene carries a different resistance gene, suitably a gene for sulphonamide resistance, for example. Increased starch production in the potato tuber would be of benefit, for example, to the potato crisp manufacturer as a 1% increase in potato dry matter would result in a 4% increase in product.
Potato crisp manufacture also serves to illustrate another benefit of the invention. When potato tubers are stored at temperatures below 8xc2x0 C., reducing sugars, glucose and fructose from the breakdown of starch accumulate. When the potatoes are fried for crisps the reducing sugars react with amino acid in the Maillard reaction to give rise to brown colouration and off-tastes in the product. Introduction into potato plants of a chimaeric gene which would stop the breakdown of starch and thus the accumulation of reducing sugars would be of benefit to the snack food industry. Preferably the chimaeric gene would comprise the coding sequence, or a part of the sequence, for ct xcex2-amylase in a cosuppression or antisense construct, driven by a suitable promoter and terminator. A suitable promoter would be taken from the gene for patatin in potato tubers. Advantageously any of the other starch degrading enzymes mentioned above could also be used instead of the ct xcex2-amylase.
The inducible promoter of SEQ. ID. No. 8 could also be used in the construct if co-ordinated expression in the developing leaf and in the developing tuber were required, as the patatin promoter is also sucrose inducible (Rocha-Sosa et al (1989). Similarly, the sequence for the chloroplast targeting polypeptide of SEQ. ID. No. 1 could also be used with any other gene which lacked its own targeting sequence and which was required to be directed to plastids.
The above examples serve to illustrate the possible benefits of using the present invention. One skilled in the art will recognise that the combination of genes and the plants to which the invention could be applied is considerable.
Gene combinations preferably will include ct xcex2-amylase with one or more of the genes for sucrose synthase, ADPG pyrophosphorylase, starch synthase, branching enzyme, xcex1-amylase, isoamylase, non-plastidic xcex2-amylase, xcex1-glucosidase, starch phosphorylase and disproportionating enzyme, the sequences of which are known to the skilled man. Alternatively, the targeting sequence from ct xcex2-amylase may be used with one or more of the above genes.
The list of plants which could be transformed preferably include potato, wheat, maize, barley, tomato, rice, pea, soybean, peanut, cassava, yam, banana and tobacco.
The invention will now be described, by way of example, with reference to an embodiment for isolation of the cDNA for ct xcex2-amylase from Arabidopsis thaliana and for incorporating the cDNA into tobacco and potato plants. Examples are also given on the stimulus responsive promoter and its activity in transgenic plants.